1. Field Of The Invention
The present invention relates to an apparatus for the sequential degradation of proteins or polypeptides, and more particularly, to an improved reaction cell for containing small volumes of sample, and for greater control of the reactions therewithin. The reaction cell can be used for other biochemical processes as well.
2. Art Background
In 1957, P. Edman published a paper which began a new era in the field of protein sequencing, enabling eventually the automated sequencing of proteins and peptides in a liquid phase. P. Edman, Acta. Chem. Scand. 10,761(1957).
The three step process first involves coupling the N-terminal amino acid of a starting polypeptide to phenylisothiocyanate (PITC) in the presence of an acid scavenger in an alkaline aqueous or anhydrous solvent. The volatile excess reagent is removed in vacuo (homogeneous phase reaction) or by washing a polypeptide immobilized on an insoluble support (heterogeneous phase reaction) and byproducts of the reaction resulting from the decomposition of PITC are similarly removed, to yield the phenylthiocarbamyl (PTC) polypeptide.
In the second step, the PTC polypeptide is subjected to cleavage by a volatile anhydrous acid to afford the 2-anilino-5(4H)-thiazolinone (ATZ) of the N-terminal amino acid and the salt of the residual polypeptide, which is the starting polypeptide with the N-terminal amino acid removed.
The ATZ amino acid is extracted from the residual polypeptide or washed from the insoluble support in the cleavage acid which is subsequently removed in vacuo or evaporated under a stream of nitrogen gas at elevated temperature. In the third step, the ATZ amino acid, is ordinarily subjected to conversion to the more stable phenylthiohydantoin (PTH) amino acid. The conversion reaction can be effected thermally or by heating the ATZ amino acid in aqueous, methanolic or anhydrous acid.
The resultant PTH amino acid is identified chromatographically while the shortened residual polypeptide is neutralized and then reacted with PITC to initiate the next cycle of degradation. The foregoing steps (coupling, cleavage and conversion) are repeated for each N-terminal amino acid in the polypeptide.
In 1967 Edman taught in, Edman et al., "A Protein Sequenator" European J.Biochem. 1 (1967) 80-91 a "spinning cup" automated sequencer which permitted the automated sequencing of peptides, still using a liquid phase separation.
In the late 1960's and early 1970's, a modification of the basic Edman chemistry was developed which involved the concepts of Edman degradation as applied using solid phase chemistry. These developments, pioneered by Richard Laursen, Eur.J. Biochem 20 (1971) 89-102 "Solid Phase Edman Degradation-An Automatic Peptide Sequencer" enabled researchers to bind the protein or peptide to be sequenced onto a solid resin, and to pump the Edman reagents past the bound peptide to provide the chemistry that sequentially degraded the peptide.
Further developments to this process were made by Dreyer, U.S. Pat. No. 4,065,412 which disclosed the use of macroporous beads to which a sample of protein or peptide is bound by chemical coupling or direct adsorption, the beads being disposed within a reaction column.
Hood, et al. in U.S. Pat. Nos. 4,252,769, 4,603,114 and 4,704,256 discloses methods and apparatus for sequential degradation of proteins and peptides by binding the samples onto a solid matrix of fluid permeable material located in a reaction chamber and applying a pressurized stream of chemicals sequentially to perform the Edman chemistry.
The emphasis at the present time is in the development of chemistries and apparatus which is substantially more sensitive, faster and in some instances, less expensive than prior art systems. The first parameter of sensitivity is particularly critical since many of the prior art methods are not entirely satisfactory in the microsequencing of small amounts of protein.
Many physiologically active proteins are present in organisms at such extremely small concentrations that only very small amounts of the proteins can be obtained for sequencing analysis. The current techniques described in the literature are aiming to obtain sensitive detecting in the picomole range. Hunkapiller and Hood, "Protein Sequence Analysis: Automated Microsequencing", Science 219 (1983) 650-659 teaches automated sequencing in the 5 to 10 picomole range.
In 1985, Hawke, Harris, and Shively, "Microsequence Analysis of Peptides and Proteins V. Design and Performance of a Novel Gas-Liquid-Solid Phase Instrument" Analytical Biochemistry 147 315-330 (1985) published an article describing an apparatus for microsequencing. This device was taken the next step by Calaycay, Rusnak and Shively, "Microsequence Analysis of Peptides and Proteins-IX. An Improved Compact Automated Instrument" Analytical Biochemistry 192 23-31 (1991) which teaches a sequencer with a sensitivity in the same range (5-10 picomoles), and a valve which is useful for microsequencing. The sequencer has a vertical flow path and a continuous flow reactor and includes a hexagonal valve for six fluid inputs to the reactor which allows sample, reagent and solvent input into a conversion flask containing two inputs on top and a drain on the bottom, the drain connecting to a valve which can connect the flask to either a waste flask or an on-line HPLC for the analysis of the PTH amino acid derivatives.
Most recently, several companies have introduced sequencers intended to handle the requirements of microsequencing, including Applied Biosystems (Protein/Peptide Sequencer PTH Analyzer 477A/120A, 473 and 476) and Milligen/ Biosearch Div. of Millipore (Prosequencer.TM.). Notwithstanding these efforts, successful consistent sequencing in the femtomole range has not yet been achieved. Moreover, the cost of these prior art devices remains substantial, making such devices unattainable for smaller laboratories.
A recent development in sequencer reaction chambers has been described by Fischer in his EPO patent No. 526691 issued February 1993. The reaction chamber is in the form of a shallow recess with an inlet and outlet at each end of the recess. The recess houses a filter element which has a sample absorbed thereon, and the reagents flow from the inlet, through or across the filter and out of the outlet. The Fischer cross-flow reaction chamber has also been described in Method of Protein Sequence Analysis, "Direct Microsequencing of Blotted and Covalently Attached Proteins in a Cross-Flow Reaction Chamber," Reinke, et al., 1991, Birkhauser Verlag Basel.
Another current reaction vessel is the type used in Applied Biosystem sequencers described in "A Modified Reaction Cartridge for Direct Protein Sequencing on Polymeric Membranes", Sheer, et al., Biotechniques, Vol. 11, No. 4, p. 526 et seq. This article describes Applied Biosystem's vertical cross-flow reaction cartridge for sequencing, made of two mating cylindrical blocks of glass with a bore through the central axis of each, and a recess in the lower block to hold a membrane. The volume of this reaction vessel can be as small as 120 microliters.
The present invention attempts to overcome some of the problems with the prior art devices in providing a reaction cell for microsequencing having a substantially smaller volume, and particularly a small dead volume which enables the sequencing of smaller amounts of protein or polypeptides. The invention can also be used to sequence DNA/RNA, and can be used in synthetic processes as well without substantial modification.